L14 - EPEC & enteric disease

Question 1

What is EPEC?

Answer 1

Enteropathogenic E. coli (EPEC)

Major cause of watery diarrhoea in infants (mostly <6-month-old)

Its primarily now a problem for developing countries – main issue due to hygiene

Question 2

What is EPEC a member of?

Answer 2

These bacteria are a member of the attachment & effacing family of pathogens – they cause changes on the cells, the attaching & facement of the absorbance to microvilli

Question 3

How does EPEC infect host cells?

Answer 3

• The bacteria target the absorbent epithelia of the small intestine
• Most bacteria cannot get through this – however EPEC can sink into the brush border of microvilli
• Make contact with the host plasma membrane
• This triggers signaling responses in the host cell that lead to the formation of pedestal-like structures
• Also associated with the elongation of the microvilli and the loss of microvilli which is thought to allow more bacteria to be recruited to the site of infection

Question 4

What is virulence of EPEC mainly dependent on?

Answer 4

The key determinant is a secretory system – the ability to be able to transfer proteins outside or into host cells is important in the disease process

They do this by a T3SS (type 3 Secretion system) – which is a molecular needle syringe like structure which connects the cytoplasm of the host and bacteria

A novelty of the EPEC is that the needle is extended by one of the secretory proteins (EspA) polymerizing to make a long extension

Question 5

Where is T3SS encoded?

Answer 5

On the Locus of Enterocyte Effacement (LEE) region

LEE is a pathogenicity island

Question 6

Structure of T3SS

Answer 6

LEE has about 40 proteins - half of them form the T3SS

Some of the proteins are secreted – secreted translocator proteins – they form the extension of the T3SS – allows the system to interact with the host plasma membrane – inserts 2 proteins into the host plasma membrane to generate a pore – allows effector proteins to be delivered into the host cell – these drive the disease process

Known that the protein in the plasma membrane (EspB) also has effector functions

There are other proteins involved in the system – regulators & chaperones

Intimin is an outer membrane protein that is critical for disease

Question 7

How did EPEC become pathogenic?

Answer 7

EPEC pathogenesis driven by acquisition of new DNA

Question 8

How can you treat EPEC diseases?

Answer 8

Can treat EPEC diseases quite easily

BUT
Rapid acquisition of antibiotic resistance by the transferring of plasmids

Question 9

What are Non-LEE-encoded (Nle) effectors?

Answer 9

While some secreted effector proteins are encoded on a pathogenicity island known as LEE, several effector molecules are encoded outside the LEE (Nle)

Has been found that a subset of these have other effector molecules that are delivered by the T3SS

They have 17 Nle effector molecules

Bacteria can deliver 24 effector molecules into the host cell

Gives it a huge capacity to alter host cellular processes

Question 10

In vitro Model of EPEC’s Target Cell: Absorptive epithelial (Enterocytes)

Answer 10

Important that studies are done of the cell types that the bacteria actually affect – need the key physiology of the target cell

Caco2 model has an insert which has a placid membrane with little holes in – not big enough for cells to fall out but it allows them to feed from both sides

Question 11

How do you get Caco 2 model cells?

Answer 11

Can take these cells from a cancer – been found you can grow them for 2/3 weeks and they spontaneously differentiate into cells that morphologically & physiologically mimics EPECs in vivo target

Question 12

How does the Caco 2 model work?

Answer 12

• Can add the bacteria to the top compartment (apical chamber) – equivalent to the lumen of the gastrointestinal tract & the bottom is equivalent to the blood system
• Can scan the surface of the cells – can see bacteria bound to the membrane
• Can see the elongation of the microvilli
• Areas where there’s no microvilli as its been removed (faced)
• In later timepoints there is lots of bacteria colonies & no microvilli

Question 13

How can you use a voltmeter in the Caco 2 model?

Answer 13

Can also use a voltmeter to look at the ability of current to travel to one compartment to the next – reflects the tightness of the cells

• If the cells are tight together the current is very slow
• If they’re disrupted the current moves
• When you effect EPEC you get rapid loss of electrical resistance
• Bacteria can disrupt these tight junctions

Question 14

Diarrhoeal-associated alteration of enterocytes

Answer 14

LEE-encoded effector activity linked to diarrhoeal-associated alterations

Can be done by 3 mechanisms:
• Alter activity of transporters in the microvilli
• Remove absorbed microvilli
• Disrupt tight junctions between cells

Question 15

LEE-encoded effector activity linked to diarrhoeal-associated alterations

Mechanism:
Alter activity of transporters in the microvilli

Answer 15

Cause them to export more ions or inhibit their ability to take up ions – to do with water balance

Bacteria alter the transporter in the apical membrane to explain the rapid onset of diarrhea

A/E lesion formation is linked to loss of major water transporter from absorptive microvilli

Question 16

LEE-encoded effector activity linked to diarrhoeal-associated alterations

Mechanism:
Remove absorbed microvilli

Answer 16

This reduces SA of the GI tract & remove the transporters – promotes diarrhoea & malnutrition

Question 17

LEE-encoded effector activity linked to diarrhoeal-associated alterations

Mechanism:
Disrupt tight junctions between cells

Answer 17

Allows the unregulated movement of ions & antigens between the cells – if they can move in an unregulated manner then water can follow it and promote diarrhoeal disease

Question 18

What does EPEC disease depend on?

Answer 18

Intimin & Tir (transmembrane intimin receptor) are critical for the disease process

Bacteria understand the host physiology & have exploited it by stealing concepts – bacteria delivers its own receptor

Question 19

How have bacteria stolen the receptor-ligand concept from Intimin-Tir association?

Answer 19

The Tir effector molecules expressed in the bacterial cytoplasm & transferred via the T3SS into the host cytoplasm where it recognises the host kinases which modify specific residues – associated with Tir being inserted into the host plasma membrane in this hairpin like structure

The middle bit is a binding site for the surface protein intimin – once inserted it is recognised by host tyrosine kinases which phosphorylate specific tyrosine residues

Following interaction, it causes clustering of the Tir receptor leading to phosphorylation of the tyrosine’s causing a signalling cascade – recruits host protein called Nik (adaptor molecule) whose function it to recruit N-WASP which activates the actin nucleating machinery called Arp2/3 which leads to these pedestal-like structures underneath the bacteria

This demonstrates how the bacteria understands the host cell physiology
• Exploit host kinases to trigger its signalling cascades to promotes its life cycle

Question 20

How is EPEC disease dependent on Tir-Intimin (receptor-ligand) interaction

Answer 20

A lot of Tirs ability to alter host cellular processes depends on it binding intimin – triggers a signaling cascade

Bacteria from outside the cell, can keep control of its effector molecules through the Tir-Intimin interaction

They allow the bacteria to sink through the absorbed microvilli – if they can’t do this they can’t deliver their effector molecules in the cytoplasm

Question 21

What is microvilli size dependent on?

Answer 21

Threadmilling

Where actin is added & polymerized at the tip, the rate of adding vs the rate of natural turnover at the other end is threadmilling – can be altered by promoting removal or addition of actin

Question 22

What proteins do many pathogens target?

Answer 22

Small RhoGTPases are a key target of many pathogens as they play a central role in controlling different signaling cascades that the bacteria manipulate

Question 23

What is MAP?

Answer 23

Map is a Cdc42 specific GEF – molecular ON/OFF switches

Cdc42 is a member of the small RhoGTPase family of molecular on-off switches of signaling cascades

When receptor signaling occurs, triggers signaling cascade that activates small GTPase via GEF and then proteins like GAF switch them off again

Question 24

What is EspF?

Answer 24

EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC)

Question 25

How do EspF & MAP share a common target?

Answer 25

• Structural studies have shown that Map is GEF – it functions specifically to activate Cdc42 which triggers the signaling cascade leading to these filopodia formation
• It’s also been found that EspF directly activates N-WASP as it has 3 poly proline rich regions – each one has an N-WASP binding domain – this recruits N-WASP
• Found that the feature is very identical to the auto-inhibitory domain in N-WASP, it binds & opens N-WASP to activate it – makes it constitutionally active to drive actin nucleation events
• Also found that N-WASP has recruitment sites for a protein called SNX9 which is linked the recruitment of the endocytic machinery to internalize the membrane
• Illistrates the knowledge of the bacteria

Question 26

Rapid onset of watery bacteria

Answer 26

Tir, Intimin, Map and EspF cooperativity removes major water pump from intact microvilli

The rapid onset of watery diarrhoea is related to the SGLT1

Question 27

How does EPEC remove SGLT1?

Answer 27

• When you infect with EPEC there is a rapid loss of SGLT1 from the apical surface
• Effector molecules work together to remove SGLT1 from the surface
• We think EspF is recruiting SNX9 in N-WASP to drive endocytosis - & the other effector molecules must be part of this process
• We know that the EspG LEE effector can prevents secretory pathways – prevents new SGLT1s being added to the microvilli
• This causes the rapid onset of watery diarrhea

Question 28

Paradox in EPEC human disease

Answer 28

• EPEC disrupts cell-cell junctions in vitro and in vivo models – this allows antigens such as flagellin (key stimuluses of inflammation) to get between the cells & interact with its TLR, TLR5
• This then triggers signalling cascades that leads to the activation of transcription factors to trigger the expression of cytokines such as IL-8 which act as alarm calls to recruit PMNs to the site of infection so they can start attacking the bacteria
• These should lead to the release of more chemokines which leads to an inflammatory diarhhoeal disease
• EPEC diseases are not associated with a strong inflammatory response which suggests there is a suppressive mechanism

Question 29

Mechanisms employed by EPEC to inhibit infection-induced gene transcriptional responses

Answer 29

NleC was found to interact with NF-kB in the nucleus and was able to cleave it as it is a metalloprotease

The complex was recruited by docking at the NleC and then has a catalytic domain that cleaves NF-kB to stop it being functional

Loss of IκB and NF-κB Rel proteins p65 and p50

When they looked at the crystal structure of NleC, it mimics the target of NF-kB

Again illustrates how bugs understand the physiology & can mimic the DNA

Question 30

EPEC Interferes with the activity of the NF-кB cofactor RPS3 by multiple mechanisms

Answer 30

• RPS3 directs NF-kB to specific genes
• Its been found that to the effector molecules, that the pathogens look like serine-threonine kinases – have stolen the structure & put a signal sequence on it & delivered it back into the host to utilize it against the host
• Found that the molecule interferes with the function of ribosome binding protein S3 although doesn’t require kinase activity
• Also found that NleH could interfere with the function of RPS3
• Illustrates that bacteria have the ability to alter specific proteins that hinder the replication pathway

Question 31

EPEC Deploys the protease strategy to inhibit Map Kinase Signalling Pathways

Answer 31

They can target other effector molecules that cleave other molecules

NleCs will go and cleave JNK & p38 – because they recognize the loop structure – a residue difference in ERK protects ERK from being degraded

However, ERK is stopped by other effector molecules

Question 32

Other Targets on the Receptor-IKK-NFкB activation pathway: NleE

Answer 32

Another target molecule targets the gene upstream from that – TAB proteins

They are important for activating the kinase

NleE catalyses the cysteine methylation of these 2 TAB proteins – prevents capacity of proteins to recruit TAK1

Question 33

Other Targets on the Receptor-IKK-NFкB activation pathway: Tir

Answer 33

Another effector molecule that targets the upstream protein TRAF6

Tir can target these proteins & lead to their degradation and other TRAFs which are associated with cytokine signaling

Tir also has other functions to inhibit the pathway – some residues on the C-terminus that are phosphorylating by the host & act as an ITIM, which recruits phosphatase to inhibit TRAF function so it doesn’t get ubiquitinated so cannot recruit the TABs – inhibits NF-kB signaling

Question 34

Other Targets on the Receptor-IKK-NFкB activation pathway: NleB

Answer 34

NleB targets proteins quite high in the pathway

Can inhibit signaling though receptor kinases but not through antigens like the TLRs suggesting its targeting something upstream of these pathways

NleB targets a specific component upstream, causing the modification of these domains – TRA domains

Adds a sugar onto arginine (novel modification) on the TRA domain which inhibits its activity

The target arginine is conserved in multiple proteins involved in regulating cell death pathways

Question 35

Other Targets on the Receptor-IKK-NFкB activation pathway: EspL

Answer 35

EspL is a cysteine protease effector molecule that targets proteins with the rim motif

Found in lots of different molecules & important in signaling through NF-kB & also protects the cell death signaling pathways

Question 36

How do bacteria survive without killing the host?

Answer 36

if you over inhibit one pathway, the cell recognizes that and causes dysfunction leading to cell death
• The bacteria understand this
• They target lots of proteins at low levels to inhibit their activity a little bit – doesn’t impact any individual step but collectively they inhibit signaling to NF-kB
• The levels of delivery are critical
• Also gives the bacteria evolutionary advantages

No pathogen wants to kill its host, it just wants to have a maximal level where it can propagate before its killed by the cell

Can lead to oversignalling & death if you’re immunocompromised or if the pathogen is extra virulent